[CANCER RESEARCH 36, 4570-4576, December 1976]

biochemical marker of malignant cells. The form that hasbeen most cleanly associated with cancer is the placentalisozyma of alkaline phosphatasa. This isozyma is heat stabile and has bean referred to as the Ragan enzyme whenfound in pathological circumstances. Thus fan, wheneverfound in any appreciable liter in nonpmagnant states, placenlal alkaline phosphatasa expression indicates a pathological condition (7, 20, 21, 28, 34, 36). Whether placentalalkaline phosphatase is a normal constituent of chomiocancinoma becauseof its trophoblashicorigin onwhether it mapresentsa deneprassadfunction as in other typesof cancer isat present unknown. Placental alkaline phosphatase hasbeen studied in a number of human cell culture lines, andits activity can be increased by glucocorticoids (5, 12, 13,29), substrates (6), butyrala (10), and hypanosmolamity (12,13, 30). The increase in placental alkaline phosphatase aclivity by hyperosmolanity is particularly intriguing, since hyperosmolanity can also inhibit DNA synthesis (38).

This paper examines the effect of MTX upon HCG produclion and placental alkaline phosphalase activity in the BeWoline of choniocarcinoma cells and demonstrates that MTX,in doses that inhibit DNA synthesis, stimulates both HCGsynthesis and placental alkaline phosphatase activity.

MATERIALS AND METHODS

Media and Chemicals. Waymouth's 752/1 medium andGey's balanced salt solution were purchased from GrandIsland Biological Co., Grand Island, N. Y. Calcium- andmagnesium-free Earle's balanced salt solution was pmapared by the NIH media unit. Fetal bovine serum was punchased from Colorado Serum Company Laboratories, Denyen, Cob. , and from North American Biologicals, Inc.,Miami, Fla. Newborn calf serum was from Grand IslandBiological Co. Lyophilizad trypsin (250 units/mg) was punchased from Worthington Biochemical Corp., Freehold,N. J. EDTA was from Schwarz/Mann, Orangabung, N. Y.MTX (sodium salt) was from Laderla Laboratories, Divisionof AmericanCyanamidCo., PearlRiven,N. Y. N-5-Fommyltatrahydnofolate (folinic acid) was from Grand Island Biological Co. Thymidina, hydroxyurea, cyhosina arabinosida, cycboheximide, actinomycin D, disodium p-nitnophenyl phosphahe, p-nilrophanol standard , 2-ammno-2-malhyl-1-propanol buffer, pyruvate, and NADH were from Sigma Chemical Co., St. Louis, Mo. 1251-labaladHCG (90 to 100 @Ci/@g),L-[4,5-3Hjleucina (60 Ci/mmole), [6-3H]unidine (24.2 Ci/

4570 CANCERRESEARCHVOL. 36

The Stimulation by Methotrexate of Human ChorionicGonadotropin and Placental Alkaline Phosphatase inCultured Choriocarcinoma Cells

K. V. Speeg, Jr.,1 Jane Clifford Azizkhan, and Kurt Stromberg

GeneRegulationSection,Laboratoryof MolecularBiology,Divisionof CancerBiologyandDiagnosis,NationalCancerInstitute,Bethesda,Maryland200142

SUMMARY

Treatment of the BeWo line of choniocancinoma cells withmethotnaxate in doses that inhibit DNA synthesis causes atenfold increase in synthesis of human chomionic gonadotropin and a threefold increase in activity of placental alkaline phosphatase. No concomitant increase in lactic dehydmogenasa activity occurs under these conditions. This effact of methotrexata can be blocked by simultaneous addilion of thymidina or folinic acid, neither of which aloneincreases human chomionic gonadotmopin synthesis or placental alkaline phosphalasa activity in BaWo cells.

INTRODUCTION

The fall in HCG3 during the course of chemotherapy forgestational choniocarcinoma is 1 of the oldest and, perhaps,the most specific example of using a biochemical marker asan index of tumor call destruction (1). As such it representsa significant achievement in chemotherapy by offering anassessment of clinical progress and an end point of chamotherapy. It was observed, however, that early in the courseof chemotherapy with MTX and/or actinomycin D, a Iransient rise occurred in the serum level of HCG (1, 18). Thishas bean attributed to release of HCG from lysad choniocarcinoma cells (1). Hussa at a!. (18) demonstrated that aclinomycin D added to a continuous line of chomiocarcinomacells in culture (BeWo line) resulted in increased amounts ofimmunopnacipitabla HCG in the cells themselves and intheir cell culture media. However, themewas far more HCGthan could be accounted for on the basis of call lysis alone.Vary few details are known about the synthesis, storage, ifany, and release of HCG by either the Irophoblast calls ofnormal placenta on chomiocancinoma calls. However, because no intracellular storage form of HCG has been deladed, it is unlikely that normal tmophoblast or chomiocarcinoma calls stoma any appreciable amount of HCG. Monaoven, the mechanism of increased HCG synthesis in choniocarcinoma calls following exposure to actinomycin D memains unknown.

Alkaline phosphalasa has also gained prominence as a

I Present address: Department of Medicine, Vanderbilt University Medical

School, Nashville, Tenn.2 Address to which requests for reprints should be addressed.

3 The abbreviations used are: HCG, human chorionic gonadotropin; MTX,

methotrexate.Received June 25, 1976; accepted September 14, 1976.

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Stimu!ation of HCG and Alkaline Phosphatase by MTX

mmole), [5-3H]unidine (26.2 Ci/mmole), and [methyl‘4Cjthymidmne(57.1 mCi/mmola) were purchased from NewEngland Nuclear, Boston, Mass. Rabbit anti-human chonionic gonadolnopin antibody and goat anti-rabbit gammaglobulin antibody were purchased from Cappel Laboratonies,Inc., Downingtown, Pa.

Cells and Culture Technique. BeWo choniocarcinomacells were obtained from the American Type Culture Colleclion, Rockville, Md. (Catalog CCL 98). Cells were grown inFalcon plastic flasks in medium containing 50% Waymouth's 752/1 medium, 40% Gay's balanced salt solution,and 10% bovine serum. Stock cultures ware grown in thepresence of penicillin (100 units/mI) and straptomycin (100

@g/ml).BaWo has previously been grown in 10% newborncalfsemum (18, 19, 32), but wafound thatfatal bovine serumgave much batter growth with retention of hormonal properties (shortened population-doubling time, batten platingefficiency, and the added advantage of successful subcultune up to 1:10 when grown in fetal bovine serum ratherthan 1:2 or 1:3 as recommended for growth in newborn calfserum). Therefore, this report utilizes calls maintained infetalbovineserum. Cultureswere maintainedat37°inanonhumidifiad atmosphere of 5% CO2-95% air. Calls weresubculturad with 0.05% trypsin-0.02% EDTA in calcium- andmagnesium-free Earle's balanced salt solution. For the axpeniments to be described, calls were subcultured to givemonolayer cultures approximately 20 to 30% confluent. Initially, the cells received medium containing fatal bovineserum for approximately 24 hr. The cultures were thenswitched to serum-free medium (55% Waymouth's 752/1medium and 45% Gay's balanced salt solution), and madium was changed every 24 hr. Inhibitors or other agentsware then added daily in serum-free medium. The mediumof treated cultures was collected daily, centrifuged to mamove call debris, and then stored at —50°until HCG wasassayed. For assay of protein, placental alkaline phosphatase, or cellular HCG,the cell layer of appropriate cultureswas washed with phosphate-buffered 0.9% NaCIsolution,pH 7.1 , to remove debris and unattached cells. The cellsheet was then harvested by scraping with a rubber policeman and rewashad with phosphate-buffered 0.9% NaCI solution by centnifugation. The calls ware resuspended in asmall volume of phosphate-buffered 0.9% NaCI solution,disrupted by sonic disruption, and stored at —50°untilassayed for protein, placental alkaline phosphatase, andcellular HCG.

Assays. Alkaline phosphalasa (EC 3.1 .3.1) was measuredby the method of Lowry (25) using p-nitmophenyl phosphateas substrate at pH 10.3. BeWo cells contain 2 types ofalkaline phosphatasa (manuscript in preparation). One ofthese is the typical placental isozyma (placental alkalinephosphatase) which is stable at 65°.The alkaline phosphalase activity reported here is that which remains after heating the enzyme preparation for 5 mm at 65°.One unit ofplacental alkaline phosphalasa is defined as that activitywhich catalyzes the formation of 1 nmole of p-nilrophenolper mm at 37°.MTX added to the enzyme assay was found tohave no effect on activity. Lactic dehydrogenase (EC1.1.1.27) was measured in the BeWo cell sonic extract bythe method of Reeves and Fimognani (33), which follows the

oxidation of NADH to NAD at 340 nm as lactate is formedfrom pyruvala. All enzyme assays ware performed in duplicate after optimal conditions ware established for linearityin respect to incubation time and enzyme concentration.Protein was measured by the method of Lowry at a!. (26)using bovine serum albumin as a standard. HCG titans weremeasured in duplicate by radioimmunoassay (31). The assay was standardized with a standard purchased from Samono Laboratories, Inc. , Boston, Mass. , which had beenmadioimmunobogically calibrated against the 2nd Inlenna

tional Standard of HCG. Themewas no HCG detected infreshly prepared medium. MTX did not interfere with theradioimmunoassay. Increasing aliquots of BeWo cellularsonic extract on media in which BaWo had grown gaveparallel displacement curves with known HCG.

Incorporation of Radioactive Precursors. Synthesis ofDNA, RNA, and protein was estimated by incorporation oflabeled thymidine, umidine, on beucina into macromoleculesby a modification of the method of Breen and Da Vellis (4).For labeling cellular DNA and RNA, BaWo cells maintainedin serum-free media wane pulsed with 1 @Ciof [methyl‘4C]lhymidinaand 1 @tCiof [5-3Hjunidine for 3 hr. The celllayer was than washed 3 times with 5 ml of 0.9% NaCIsolution containing 4 mu unidine and 17 @Mthymidina. Thecells were then harvested by scraping with a rubber policeman in a 3-mbvolume of fresh, cold 5% tnichboroacatic acidand by centrifuging at 1000 x g for 10 mm at 4°.The supemnatant was discarded. The pallet was washed twice with a 2-mIvolume of cold 5% tnichboroacetic acid and once with a 2-mb

volume of 100% ethanol. The pellet was dissolvad,in 1.5 mlof 0.1 M KOH and heated at 100°for 30 mm to hydrolyzeRNA. The mixture was cooled, and 5 M parchlonic acid wereadded to a final volume of 2 ml. The precipitate containingDNA was collected by cantnifugation and saved. Aliquots ofthe supamnatant ware counted in Aquasol in a liquid scintillation spectrometer. The RNA content was determined fromthe absorbanca of the supemnalant at 260 nm. The palletcontaining DNA was washed twice with cold 1 N perchbonicacid. To the pellet was then added 2 ml of 0.25 NNaOH, andthis was healed at 100°for 10 mm. The solubilizad DNA wascooled to room temperature, and the DNA content wasdetermined from the absonbance at 260 nm. Radioactivitywas measuredby liquid scintillation spactrometmyin Aquasob. This method gave 95% separation of DNA and RNAbased on separation of 3H and 14Cradioactivity. The abovemethod was used to assess the inhibition by actinomycin D.The method was also used to assess the inhibition by MTX,except that [6-3H]umidine was incorporated into DNA andRNA instead of Ihymidina and [5-3H]unidine. Labeling ofcellular protein was by the method of Breen and Da VeIlis(4).

RESULTS

Stimulation of Placental Alkaline Phosphatase MTX.Chart 1 illustrates the effect of adding 1 @MMTX for 24 hr tochoniocarcinoma cells (BeWo) grown in defined medium incell culture. Cell growth is arrested after the addition of 1,.@MMTX, and this effect persists for approximately 2 daysafter removal of MTX (Chart 1E). Call growth, as measured

4571DECEMBER1976

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by increase in call protein (14), than resumes at the samemateas in the control flasks. Themeis an increase in totalcellular placental alkaline phosphalasa activity in the MTXtreated flasks in comparison to untreated control flasks(Chant 1A). In Chart 1B, the placental alkaline phosphataseactivity pen mg cell protein (placental alkaline phosphahasespecific activity) is seen to have a lag period of approximately 1 day followed by a more rapid increase for 2 days.This is then followed by a return in specific activity of placental alkaline phosphatasa to levels approaching those ofthe control flasks. The average (±S.E.) increase of specificactivity of placental alkaline phosphatase observed 72 hrafter addition of 1 p@MMTX was 295 ±35% (range, 220 to410% increase).

As shown in Chart 1, C and D, HCG production andsecretionintothe medium inuntreatedcontrolflasksdadma ho a baseline level after subculture and removal of

K. V. Speag, Jr. , at a!.

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serum. If BeWo calls are treated with 1 @MMTX, the amountof HCG released into the medium rises after a 1-day lagperiod and persists for about 2 days after removal of MTX.The level than falls toward that of untreated cells. Thesechanges in HCG found in the media are also reflected bycellular HCG (Chart iF), except that the cellular HCG penmgcell protein is 50- to 100-fold lass than that found in themedia. The average amount (±S.E.) of stimulation of HCGsynthesis seen with 1 @MMTX 72 hr after addition of thedrug was 980 ±250% (range, 360 to 2300% increase). Thechanges in placental alkaline phosphatase activity or HCGcontent correlate closely with the inhibition of cell growthcaused by a 24-hr exposure to MTX and the resumption ofcell growth 2 to 3 days after the removal of MTX, as showninChart 1E.

Effect of MTX on DNA, RNA, and Protein Synthesis. Theeffect of various concentrations of MTXon DNA, RNA,andprotein synthesis in BeWo cells grown without serum isshown in Table 1. DNA synthesis was inhibited 90 ho 95%with either 0.1 on 1 @MMTX. In contrast, 0.01 @MMTX hadessentially no effect on DNA synthesis. None of these concentrations of MTX inhibited RNA synthesis even after 72 hrin the presence of the drug. MTX inhibited protein synthesisto a moderate extent. Cycboheximide (3.5 @M)is included asa referencefor inhibition of protein synthesis.Notethat thisconcentration of cycioheximida inhibited protein synthesisapproximately 85% initially, but, subsequently, its effectdiminished. Actinomycin D (5 nM) inhibited RNA synthesisapproximately 60% and reduced DNA synthesis somewhat

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The change in specific activity of placental alkaline phosphatase and HCG with increasing doses of MTX is shown inChart 2. Exposure of BeWo cells to 0.01 @&MMTX over a 72-hr period results in placental alkaline phosphalase activitysimilar ho that of untreated cells (Chart 2,4). However, MTXdoses between 0.1 and I 0 @Mcause an increase in placentalalkaline phosphalase activity that becomes more dose rebated late in the experiment. There is no response in HCGproduction with 0.01 @MMTX, but an increase occurs withdoses between 0.1 and 10 @M(Chant 28). In contrast to thechange in placental alkaline phosphatase activity, the increasein HCGproduction appearsto be dose relatedearlierin the experiment. These results agree well with those presenled in Table 1, where 0.01 @MMTX caused no inhibitionof DNA or RNA synthesis, but higher doses resulted insignificant inhibition of DNA synthesis. It should be pointedout that the “response―depicted here is cleanly a complexone involving transport of MTX into the cell, inhibition ofdihydrofolate reductase, a probable accumulation of unbound MTX, a decrease in available thymidine, an inhibitionof DNA synthesis (3, 8, 11, 15, 16, 37), and, ultimately, anincrease in HCG and placental alkaline phosphatase specific activity by an unknown mechanism(s). Superimposedon this is a possible attempt by the cell to synthesize newdihydrofolateneductasaand reversetheinhibitionofDNAsynthesis caused by a lack of thymidina (11, 15, 37).

Lack of Stimulation of Lactic Dehydrogenase. To ascerlain whether MTX might be increasing other choniocarcinoma functions in a nonspecific way, the cellular lactic

Chart 1. Effect of MTX upon HCG and placental alkaline phosphatase inBeWo cells. Cells were subcultured on Day 0 and fed serum-free medium oneach subsequent day. One @MMTX ( 0) in serum-free medium or serum-freemedium without MTX (C) was added on Day 3 and removed on Day 4 (i). Atthe indicated times, media and cells were collected for HCG and placentalalkaline phosphatase, respectively. A , activity of placental alkaline phosphatase (PAP) per culture in MTX-treated and untreated cultures; B, placentalalkaline phosphatase (PAP) measured as in A, expressed as specific activity;C, amount of HCG secreted into the culture medium per 24 hr by treated anduntreated cultures; D, amount of HCG per 24 hr as in C, corrected for amountof cell protein; E, effect of MTX on cell growth as measured by increasingamount of cell protein with time in culture; F, amount of cellular HCG per mgcellular protein in MTx-treated and untreated cultures.

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Stimulation of HCG and Alkaline Phosphatasa by MTX

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Chart 2. Dose-response stimulation of BeWo placental alkaline phosphatase and HCG by MTx. Cells were subcultured on Day 0 and fed serum-freemedium on each subsequent day. On Day 3, MTX was added in serum-freemedium for the 72 hr indicated (). At the indicated times, cells werecollected for assay of specific activity of placental alkaline phosphatase(PAP)(A)or assayof the amountof HCGsecretedinto the culture mediumper 24 hr expressed per mg cellular protein (B). Doses of MTx: none (•),10nM (0), 0.1 gLM(A), 1 @M(0), 10 @M(•).

dehydmogenase was measured at the same time-points andMTX concentrations. The lactic dehydmogenase specific activily was found to be unchanged in control and MTXtreatedcells.

Use of Other Inhibitors. Attempts at stimulation of thelevel of HCG production and placental alkaline phosphatasaactivity with other inhibitors of DNA synthesis (35) amashown in Chart 3. Cytosina arabinoside at 1 @Mcaused a 2-fold increase in placental alkaline phosphalase activity anda 4-fold increase in HCG production. A less dramatic shimulation of HCG synthesis and, probably, no stimulation ofplacental alkaline phosphatase activity occurred followingexposure to 1 ,.@Mhydroxyunea. Further study would berequired to demonstrate that either of these drugs is capabla of inhibiting DNA synthesis in BaWo calls, specifically,and what might be the optimal conditions for stimulation.

Effect of Thymidine and Folinic Acid. BaWo calls werefound to grow well in hypoxanthine-aminoptenin-thymidinamedium. This presumably reflects the presence in thesecells of thymidina kinase and hypoxanthine-guanine phosphonibosyltransfarase (23, 24). Because Waymouth's 752/1medium is relatively rich in hypoxanthine, the increase inHCG synthesis and placental alkaline phosphatase activityafter exposure to MTX was considered to be secondary todeprivation of thymidine and subsequent inhibition of DNAsynthesis. Because MTX is a potent inhibitor of dihydrofolate maductase,which is essential for biosynthesis of thymidine, several mixing experiments with MTX, thymidina, andfolinic acid were performed. Chart 4 illustrates that MTXalone results in an increase in HCG and placental alkalinephosphahase. However, thymidine alone on the simultaneous addition of MTX and excess Ihymidine did not result in

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Chart 3. Effect of other inhibitors on HCG and placental alkaline phosphatase in BeWo cells. Cells were subcultured on Day 0 and fed serum-freemedium on each subsequent day. On Day 3, 1 @iMhydroxyurea (A , C) or 1 @.tMcytosine arabinoside (B, D) was added for the indicated time (). At appropriate times, cells treated with inhibitor ( 0) or cells grown without inhibitor (•)were harvested for placental alkaline phosphatase specific activity orthe amount of HCG secreted into the culture medium per 24 hr expressed permg cellular protein.

DISCUSSION

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Chart 4. Effect of thymidine on stimulation of BeWo HCG and placentalalkaline phosphatase by MTX. Cells were subcultured on Day 0 and fedserum-free medium on each subsequent day. On Day 3, 1 @MMTX (ti), 1 @MMTX plus 0.1 mM thymidine (), 0.1 mM thymidine (S), or no addition( 0) in serum-free medium was added for the indicated time (). At appropriate times, cells were harvested for placental alkaline phosphatase(PAP)specificactivity(top) or the amountof HCGsecretedinto the culture

. medium per 24 hr expressed per mg cellular protein (bottom).

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Chart 5. Effect of folinic acid on stimulation of BeWo HCG and placentalalkaline phosphatase by MTx. Cells were subcultured on Day 0 and fedserum-free medium on each subsequent day. On Day 3, 1 @.tMMIX ( 0), 1 @MMTX plus 0.1 mM folinic acid (La),or no addition (•)in serum-free mediumwas added for the indicated time (). At appropriate times, cells wereharvested for placental alkaline phosphatase (PAP) specific activity (top) orthe amount of HCG secreted into the culture medium per 24 hr expressed permg cellular protein (bottom).

an increase in either HCG synthesis or placental alkalinephosphatase specific activity. Moreover, as shown in CiSant5, the increase in both HCG synthesis and specific activity of

placental alkaline phosphatase can be prevented by theaddition of 0.1 mM folinic acid at the time of addition of 1

@MMTX. The folinic acid acts as a 1-carbon donor toward

the biosynthesis of thymidina and is not dependent upond ihyd rofolata red uctase activity (1 5).

The demonstration that MTX stimulates HCG synthesisand placental alkaline phosphalasa activity and that thestimulation is related to inhibition of DNA synthesis is basedon the following data: (a) the addition of MTX results in anincrease in the amount of cellular HCG and HCG secretedintothe cellculturemedium as measured by radioimmunoassay, as wall as an increase in the specific activity ofcellular placental alkaline phosphalasa; (b) it is those dosesof MTX that can be shown to inhibit DNA synthesis but thatdo not inhibit RNA synthesis that stimulated HCG synthesisand placental alkaline phosphatase activity [the inhibitionof DNA synthesis by the doses of MTX reported hare is ingood agreement with that found by Hussa and Patillo usingBaWo cells (17)]; (c) drugs (Ihymidine and folinic acid) thatcircumvent the action of MTX on DNA synthesis prevent thestimulation of HCG and placental alkaline phosphahasa. Theeffect may be a specific one because lactic dehydrogenasedoes not increase with doses of MTX which inhibit DNAsynthesis.

The assumption of an actual increase in the synthesis of

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Stimulation of HCG and Alkaline Phosphatase by MTX

HCG has been made because of the measured increase bycompetition radioimmunoassay and lack of evidence for amechanism of destroying HCG in BeWo cells. Confirmationof an increase in newly synthesized HCGafter BeWo cellsare exposed ho MTX is ultimately required by anothermethod. Induction of placental alkaline phosphatase cannot be assumed, since measurement is made of enzymeactivity and not of enzyme content. Whether the activityincreases secondary to increased synthesis, decreaseddegradation, or due to enzyme modification is at presentunknown and requires further study.

After prolonged exposure to 1 @MMTX, BaWo cells areeventually killed. Therefore, it might be argued that MTXeliminates a subpopulation of BeWo calls which is bothmore sensitive to the drug and also one which producesless HCG and a lower specific activity of placental alkalinephosphahasa. The result would be a factitious increase inspecific activity of both after MTX treatment. The data inChart 1 demonstrate that selective cell destruction by MTXis unlikely. When the total activity of placental alkalinephosphalase or content of HCG are plotted as in Chart 1, Aand C (uncorrected for the amount of cellular protein), bothfunctions are significantly elevated over control cultures.The increaseinHCG ismuch more thancan be accountedfor on the basis of cell bysis.

Wiltshaw and Moloney (40) have previously reported thatleukocyte alkaline phosphalase activity from normal humancontrols, patients with chronic myaboganous leukemia, andpatients with pyogenic infections can be stimulated withboth aminoptamin and amathophenin (MTX). MTX gave betterstimulation than aminoptenin. The stimulation of alkalinephosphatase by both drugs could be blocked by folinic acidif added with the inhibitors. Glutamine synthetasa activity inchick embryonic neural retina has been reported to bestimulated by cyhosina arabinosida, and the increase inactivity could be blocked by prior addition of actinomycin D(27). However, further study has convincingly demonstratedthat inhibition of DNA synthesis is not causally related to theinduction of glutamine synthetase (22). Concentrations ofcyhosine arabinoside that inhibit DNA synthesis ama2 to 3logs lower than those required to induce glutamine synthetase. Furthermore, other inhibitors of DNA synthesis suchas MTX, hydmoxyumaa,and fluorodeoxyumidinafailed to induce glutamine synthatase. The high dose of cytosina amabinoside apparently inhibits RNA synthesis sufficiently toblock a regulator of glutamine synthatase while allowingtranscription and translation of the enzyme (22). Wicks at a!.(39) have found that, in rat hepaloma, tyrosina aminotmansfenase and phosphoenol pyruvate carboxykinase are inducad by derivatives of cyclic 3',S'-AMP. Only those denivalives which could be shown to inhibit DNA synthesis wouldinduce the 2 enzymes. It is not clean whether the inductionof tymosineaminotransfarase and phosphoenol pyruvatecarboxykinase and the inhibition of DNA synthesis are ralated. Wheatley (38) has shown that DNA synthesis in HeLa53 is inhibited by hypenosmolanity. Nitowsky at a!. (30)found that hyperosmolamity would stimulate HaLa S3 alkaline phosphatasa. Hyperosmolanity causes both an increasein alkaline phosphatase activity and a decrease in growthrate in T24 cells (derived from a human urinary bladder

carcinoma). Themeis, however, no evidence as yet to mdicale that these phenomena are related (13).

The mechanism by which inhibition of DNA synthesismight cause an increase in HCG is completely unknown.One possibility is that transcription and/or translation mightoccur in only 1 phase of Iha cell cycle (for instance, in 0,).After synthesis of DNA is inhibited and progress through Sphase is arrested , BeWo cells would accumulate in 0, . Withan increasing proportion of the cells in G,, more transcniplion-translation would occur. This might account for therelatively small increase during the initial 24 hn, which isalso an approximation of the call cycle lime for BeWo calls.The continued presence of 1 pM MTX for approximately 24hr is also required for the increase in HCG and placental

alkaline phosphatase to occur (data not shown), eventhoughHussaand Pattilbo(17)foundthat1 @MMTX causedalmost complete inhibition of DNA synthesis within a few hrafter addition. While a simple increase in the number ofcalls in G@might explain a 2- to 3-fold increase in placentalalkaline phosphatase activity, it would not explain a 10-foldincrease in HCG. In other cell types, 0, occupies approximately 50% of the call cycle (2), and in a random cultureonly 50% of the cells might be expected to be in phasesother than G, . Alternatively, a short-lived negative regulatormade only during 02 might explain the increase in synthesisof HCG. Some data are consislent with the existence ofsuch a regulator in the HaLa alkaline phosphatasa system.Goz (9) has found that iododaoxyunidine stimulates HeLaalkaline phosphalase after a 24-hr bagperiod. This presumably occurs after iododaoxyumidina is incorporated intoDNA, since it may be blocked with excess thymidine. Oneinterpretation is that the iododeoxyunidine interferes withthe transcription of a regulator of alkaline phosphatasawhile allowing the enzyme to be formed as usual.

The previous report by Hussa at a!. (18), demonstrating astimulation of HCG by actinomycin D, is also consistent withthe increase occurring secondary to inhibition of DNA synthesis. Their study shows a good correlation between inhibition of DNA synthesis by actinomycin D and increase inHCG synthesis. This interpretation, however, is complicatedby the fact that actinomycin D inhibits RNA synthesis also.

Because HCG can be increased by MTX (which specificallyinhibits DNA but not RNA synthesis) and can also be increased by actinomycin D (which inhibits both DNA andRNA synthesis), it would appear that caution should beexercised in assuming that changes occurring secondary toadditionof actinomycinD to a system are due to inhibitionof RNA synthesis alone.

While the previously mentioned transient increase in Serum HCG of patients undergoing chemotherapy for gasta

tional choniocarcinoma probably does reflect to some axtent lysis of malignant cells, it is clean that cell lysis need not

be the only explanation. Although the use of HCG has beena reliable marker during chemotherapy of choniocarcinoma,it should be clear that a broaden understanding of the fac

tons modulating the HCG level, or the level of any tumormarker under various circumstances, is critical to theproper interpretation of the marker level. The reliability ofHCG as a marker may be due more to the fact that allchoniocancinomas make HCG (thus fan no human choniocan

4575DECEMBER 1976

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K. V. Speeg, Jr. , et a!.

18. Hussa, R. 0., Pattillo, R. A., Delfs, E., and Mattingly, R. F. Actinomycin DStimulation of HCG Production by Human Choriocarcinoma. Obstet.Gynecol., 42: 651—657,1973.

19. Hussa, R. 0., Story, M. T., and Pattillo, R. A. Regulation of HumanChorionic Gonadotropin (HCG) Secretion by Serum and Dibutyryl CyclicAMP in Malignant Trophoblast Cells in Vitro. J. Clin. Endocrinol. Metab., 40: 401-405, 1975.

20. Inglis, N. R., Kirley, S., Stolbach, L. L., and Fishman, W. H. Phenotypesof the Regan lsoenzyme and Identity Between the Placental D-Variantand the Nagao Isoenzyme. Cancer Res., 33: 1657-1661, 1973.

21. Jacoby, B. , and Bagshawe, K. D. Placental-type Alkaline Phosphatasefrom Human Tumor Tissue. Clin. Chim. Acta, 35: 473-481 , 1971.

22. Jones, R. E., and Moscona, A. A. Effects of Cytosine Arabinoside onDifferential Gene Expression in Embryonic Neural Retina. I. Accumulation of Glutamine Synthetase with Suppression of Macromolecular Synthesis. J. Cell Biol., 61: 688-700, 1974.

23. Littlefield, J. Selection of Hybrids from Mating of Fibroblastsin Vitro andTheir Presumed Recombinants. Science, 145: 709-710, 1964.

24. Littlefield, J. The Use of Drug Resistant Markers to Study the Hybridization of Mouse Fibroblasts. Exptl. Cell Rae., 41: 190-196, 1966.

25. Lowry, 0. H. Micromethods for the Assay of Enzymes. Methods Enzymol., 4: 366—372,1957.

26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. ProteinMeasurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265-275,1951.

27. Moscona, A. A. , Moscona, M., and Jones, R. E. Induction of GlutamineSynthetase in Embryonic Neural Retina in Vitro by Inhibitors of Macromolecular Synthesis. Biochem. Biophys. Res. Commun., 39: 943-949,1970.

28. Nathanson, L., and Fishman, W. H. New Observations on the Reganlsoenzyme of Alkaline Phosphatase in Cancer Patients. Cancer, 27:1388—1397,1971.

29. Nitowsky, H. M., and Herz, F. Hormonal Regulation of Alkaline Phosphatase in Dispersed Cell Cultures. Biochem. Biophys. Res. Commun., 11:261-266, 1963.

30. Nitowsky, H. M., Herz, F., and Geller, S. Induction of Alkaline Phosphatase in Dispersed Cell Cultures by Changes in Osmolarity. Biochem.Biophys. Res. Commun., 12: 293-299, 1963.

31. Odell, W. D., Ross, G. T., and Rayford, P. L. Radioimmunoassay forLuteinizing Hormone in Human Plasma or Serum: Physiological Studies.J. Clin. Invest., 46: 248-255, 1967.

32. Pattillo, R. A., and Gey, G. 0. The Establishment of a Cell Line of HumanHormone-synthesizing Trophoblastic Cells in Vitro. Cancer Res., 28:1231-1236, 1968.

33. Reeves, W. J. , Jr. , and Fimognari, G. M. L-Lactic Dehydrogenase: Heart(H4). Methods Enzymol., 9: 288-294, 1966.

34. Rosen, S. W., Weintraub, B. D., Vaitukaitus, J. L., Sussman, H. H.,Hershman, J. M., and Muggia, F. M. Placental Proteins and Their Subunits as Tumor Markers. Ann. Internal Med., 82: 71-83, 1975.

35. Scott, W. A., and Tomkins, G. M. The Use of Inhibitors in the Study ofHormone Mechanisms in Cell Culture. Methods Enzymol. , 40: 273-292,1975.

36. Stolbach, L. L. , Krant, M. J. , and Fishman, W. H. Ectopic Production ofan Alkaline Phosphatase lsoenzyme in Patients with Cancer. New EngI.J. Med., 281: 757-762, 1969.

37. Werkheiser, W. C. The Relation of Folic Acid Reductase to AminopterinToxicity. Proc. Am. Assoc. Cancer Res., 3: 161, 1960.

38. Wheatley, D. N. Cell Growth and Division in Hypertonic Medium. Exptl.Cell Res., 87: 219-232, 1974.

39. Wicks, W. D., Van Wijk, R., and McKibbin, J. B. Stimulation of EnzymeSynthesis and Inhibition of DNA Synthesis and Growth Rate by CyclicAMP Derivatives in Cultured Hepatoma Cells. Advan. Enzyme Regulation, 11: 117-135, 1973.

40. Wiltshaw, E., and Moloney, W. C. Studies on Various Factors InfluencingLeukocyte Alkaline Phosphatase Activity. J. Lab. Clin. Med., 47: 691-699, 1956.

4576 CANCER RESEARCH VOL. 36

cinoma that lacks HCG synthesis has been well documanted) rather than to a steady level of synthesis of HCG bythe choniocarcinoma cells.

ACKNOWLEDGMENTS

The authors are indebted to Dr. G. T. Ross and Dr. R. A. Pattillo for initialadvice and encouragement. The technical assistance of Patsy Trisler isgratefully acknowledged.

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1976;36:4570-4576. Cancer Res   K. V. Speeg, Jr., Jane Clifford Azizkhan and Kurt Stromberg  Choriocarcinoma CellsGonadotropin and Placental Alkaline Phosphatase in Cultured The Stimulation by Methotrexate of Human Chorionic

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